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1.
Microsomal fractions from developing shoots of adult white clover plants (of genotype AcAc) and cotyledons of dark germinated clover seedlings can synthesize 2-hydroxy-2-methylpropanenitrile and 2-hydroxy-2-methylbutanenitrile, the aglycone precursors of the cyanogenic glucosides, linamarin and lotaustralin, from various precursors in the presence of NADPH. l-Valine, 2-methylpropanal oxime, and 2-methylpropanenitrile are converted to 2-hydroxy-2-methylpropanenitrile and are detected as cyanide and acetone. l-Isoleucine and 2-methylbutanal oxime are converted to 2-hydroxy-2-methylbutanenitrile and are detected as cyanide and 2-butanone. At least two steps in these conversions are missing in microsomes from plants of genotype acac.  相似文献   

2.
Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid–derived cyanogenic glucosides (α-hydroxynitrile glucosides) by specific β-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores. We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled. Catabolic mutants could be placed in two complementation groups, one of which, cyd2, encoded the β-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related β-glucosidase, BGD4, were identified. This indicated that BGD4 plays no role in cyanogenesis in L. japonicus in vivo. Biochemical analysis confirmed that BGD4 cannot hydrolyze linamarin or lotaustralin and in L. japonicus is specific for breakdown of related hydroxynitrile glucosides, such as rhodiocyanoside A. By contrast, BGD2 can hydrolyze both cyanogenic glucosides and rhodiocyanosides. Our genetic analysis demonstrated specificity in the catabolic pathways for hydroxynitrile glucosides and implied specificity in their biosynthetic pathways as well. In addition, it has provided important tools for elucidating and potentially modifying cyanogenesis pathways in plants.  相似文献   

3.
Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic α–hydroxynitrile glucosides lotaustralin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific β–glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non‐cyanogenic γ‐ and β–hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only β–glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site‐directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non‐flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic β–glucosidase, resembling BGD2, and required no more than a single amino acid substitution.  相似文献   

4.
Zagrobelny M  Møller BL 《Phytochemistry》2011,72(13):1585-1592
Cyanogenic glucosides are important components of plant defense against generalist herbivores due to their bitter taste and the release of toxic hydrogen cyanide upon tissue disruption. Some specialized herbivores, especially insects, preferentially feed on cyanogenic plants. Such herbivores have acquired the ability to metabolize cyanogenic glucosides or to sequester them for use in their own predator defense. Burnet moths (Zygaena) sequester the cyanogenic glucosides linamarin and lotaustralin from their food plants (Fabaceae) and, in parallel, are able to carry out de novo synthesis of the very same compounds. The ratio and content of cyanogenic glucosides is tightly regulated in the different stages of the Zygaena filipendulae lifecycle and the compounds play several important roles in addition to defense. The transfer of a nuptial gift of cyanogenic glucosides during mating of Zygaena has been demonstrated as well as the possible involvement of hydrogen cyanide in male assessment and nitrogen metabolism. As the capacity to de novo synthesize cyanogenic glucosides was developed independently in plants and insects, the great similarities of the pathways between the two kingdoms indicate that cyanogenic glucosides are produced according to a universal route providing recruitment of the enzymes required. Pyrosequencing of Z. filipendulae larvae de novo synthesizing cyanogenic glucosides served to provide a set of good candidate genes, and demonstrated that the genes encoding the pathway in plants and Z. filipendulae are not closely related phylogenetically. Identification of insect genes involved in the biosynthesis and turn-over of cyanogenic glucosides will provide new insights into biological warfare as a determinant of co-evolution between plants and insects.  相似文献   

5.
Lotus japonicus was shown to contain the two nitrile glucosides rhodiocyanoside A and rhodiocyanoside D as well as the cyanogenic glucosides linamarin and lotaustralin. The content of cyanogenic and nitrile glucosides in L. japonicus depends on plant developmental stage and tissue. The cyanide potential is highest in young seedlings and in apical leaves of mature plants. Roots and seeds are acyanogenic. Biosynthetic studies using radioisotopes demonstrated that lotaustralin, rhodiocyanoside A, and rhodiocyanoside D are derived from the amino acid l-Ile, whereas linamarin is derived from Val. In silico homology searches identified two cytochromes P450 designated CYP79D3 and CYP79D4 in L. japonicus. The two cytochromes P450 are 94% identical at the amino acid level and both catalyze the conversion of Val and Ile to the corresponding aldoximes in biosynthesis of cyanogenic glucosides and nitrile glucosides in L. japonicus. CYP79D3 and CYP79D4 are differentially expressed. CYP79D3 is exclusively expressed in aerial parts and CYP79D4 in roots. Recombinantly expressed CYP79D3 and CYP79D4 in yeast cells showed higher catalytic efficiency with l-Ile as substrate than with l-Val, in agreement with lotaustralin and rhodiocyanoside A and D being the major cyanogenic and nitrile glucosides in L. japonicus. Ectopic expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in L. japonicus resulted in a 5- to 20-fold increase of linamarin content, whereas the relative amounts of lotaustralin and rhodiocyanoside A/D were unaltered.  相似文献   

6.
《Phytochemistry》1986,25(10):2299-2302
Experiments in which unlabelled and [aglycone 14C-labelled cyanogenic glycosides, linamarin and lotaustralin, were fed to larvae of the moth Zygaena trifolii on leaves of an acyanogenic strain of their food plant, Lotus corniculatus, showed that the larvae retained about 20–45% of the glucosides consumed. The larvae in nature usually feed on plants of L. corniculatus which themselves contain linamarin and lotaustralin. Earlier experiments had shown that the larvae of Zygaena spp. are able to synthesize these glucosides from valine and isoleucine and so both sequestration and biosynthesis of the same compounds can occur. This is the only such occurrence yet known in the relationships between plants and insects.  相似文献   

7.
Cyanogenesis in Trifolium repens L. is under the control oftwo loci; Ac/ac and Li/Hi control cyanogenic glucoside and linamaraseproduction respectively. Results obtained show that neitherthe dominant allele (Ac) coding for cyanogenic glucoside productionnor the dominant allele (Li) coding for linamarase productionare expressed in roots, seeds or seedlings before shoot emergence.Both linamarase and cyanogenic glucoside are produced duringshoot growth and there is little turnover of cyanogenic glucosidein mature leaves. As the leaves senesce there is breakdown ofthe mechanism separating cyanogenic glucoside and linamarase,since cyanogenic glucoside is lost in plants of genotype AcAc Li Li but not in those of genotype Ac Ac Li Li. About 60%of the cyanogenic glucoside produced was lotaustralin, in shootsof plants which were fed with equal quantities of the precursoramino acids L-valine and L-isoleucine. In contrast, the proportionof cyanogenic glucoside as lotaustralin found in leaves of oneplant, was only 40%. Different plants were shown to producedifferent quantities of cyanogenic glucoside, and the amountproduced was dependent on temperature.  相似文献   

8.
Zygaena larvae sequester the cyanogenic glucosides linamarin and lotaustralin from their food plants (Fabaceae) as well as carry out de novo biosynthesis of these compounds. In this study, Zygaena filipendulae were reared on wild-type Lotus corniculatus and wild-type and transgenic L. japonicus plants with differing content and ratios of the cyanogenic glucosides linamarin and lotaustralin and of the cyanoalkenyl glucosides rhodiocyanoside A and D. LC-MS analyses, free choice feeding experiments and developmental studies were used to examine the effect of varying content and ratios of these secondary metabolites on the feeding preferences, growth and development of Z. filipendulae. Larvae reared on cyanogenic L. corniculatus developed faster compared to larvae reared on L. japonicus although free choice feeding trials demonstrated that the latter plant source was the preferred food plant. Larvae reared on acyanogenic L. corniculatus showed decelerated development. Analysis of different life stages and tissues demonstrate that Z. filipendulae strive to maintain certain threshold content and ratios of cyanogenic glucosides regardless of the composition of the food plants. Despite this, the ratios of cyanogenic glucosides in Z. filipendulae remain partly affected by the ratio of the food plant due to the high proportion of sequestering that takes place.  相似文献   

9.
Transgenic cassava (Manihot esculenta Crantz, cv MCol22) plants with a 92% reduction in cyanogenic glucoside content in tubers and acyanogenic (<1% of wild type) leaves were obtained by RNA interference to block expression of CYP79D1 and CYP79D2, the two paralogous genes encoding the first committed enzymes in linamarin and lotaustralin synthesis. About 180 independent lines with acyanogenic (<1% of wild type) leaves were obtained. Only a few of these were depleted with respect to cyanogenic glucoside content in tubers. In agreement with this observation, girdling experiments demonstrated that cyanogenic glucosides are synthesized in the shoot apex and transported to the root, resulting in a negative concentration gradient basipetal in the plant with the concentration of cyanogenic glucosides being highest in the shoot apex and the petiole of the first unfolded leaf. Supply of nitrogen increased the cyanogenic glucoside concentration in the shoot apex. In situ polymerase chain reaction studies demonstrated that CYP79D1 and CYP79D2 were preferentially expressed in leaf mesophyll cells positioned adjacent to the epidermis. In young petioles, preferential expression was observed in the epidermis, in the two first cortex cell layers, and in the endodermis together with pericycle cells and specific parenchymatic cells around the laticifers. These data demonstrate that it is possible to drastically reduce the linamarin and lotaustralin content in cassava tubers by blockage of cyanogenic glucoside synthesis in leaves and petioles. The reduced flux to the roots of reduced nitrogen in the form of cyanogenic glucosides did not prevent tuber formation.  相似文献   

10.
The levels of cyanogenic glucosides (linamarin and lotaustralin) and the activity of linamarase were studied in 5-day old seedlings of oil flax (Linum usitatissimum L., cv. LCSD 200) under different environmental conditions. White light enhanced the cyanoglucosides content, and this effect depended on its intensity and the time of exposure. The level of cyanoglucosides rose with temperature, and it reached the highest level at the highest temperature (30 °C). Linamarase (EC. 3.2.1.21) activity was the highest at 20°C, especially in light-grown seedlings. Lower enzyme activity at the extreme temperature (15 and 30 °C) was observed. Water stress (low water potential, ω=−0.34 MPa) reduced by more than twice the cyanoglucoside level and linamarase activity. The possible protective, or/and regulatory roles of cyanogenic glucosides was discussed.  相似文献   

11.
《Insect Biochemistry》1987,17(5):689-693
14C-labelled 2-methylpropanenitrile and 2-methylbutanenitrile were administered to larvae and imagines of Heliconius melpomone and to larvae of Zygaena trifolii and the incorporation into the cyanogenic glucosides, linamarin and lotaustralin, was measured. Both species incorporated the precursors at all stages tested, at a high level of 15–72%, thereby indicating that the nitriles are probale intermediates in the lepidopteran biosynthesis of linamarin and lotaustralin from valine and isoleucine respectively.  相似文献   

12.
The burnet moth Zygaena filipendulae L. contains the cyanogenic glucosides linamarin and lotaustralin, which can be degraded to the volatiles hydrogen cyanide (HCN), acetone and 2‐butanone. Linamarin and lotaustralin are transferred from the male to female during mating and thus are considered to be involved in mating communication. Because volatile semiochemical cues play a major role in mating communication in many insect species, the emissions of HCN, acetone and 2‐butanone from Z. filipendulae are characterized in the present study, aiming to determine the interplay between the degradation products of cyanogenic glucosides and pheromones. The volatile emissions from Z. filipendulae and flowers inducing mating are measured using headspace solid‐phase micro‐extraction and gas chromatography‐mass spectrometry analysis. All Z. filipendulae life stages emit HCN, acetone and 2‐butanone. Virgin females show higher emissions than mated females, whereas mated males have higher emissions than virgin males. Hydrogen cyanide is only rarely detected in the course of male–female copulation. These observations indicate a role for the cyanogenic glucoside derived volatiles in female calling and male courtship behaviours, although not as a defence during copulation. Males rejected for mating by a female are accepted after injection of linamarin or lotaustralin, demonstrating that cyanogenic glucosides are also important for female assessment of the fitness of the male. Volatiles from flowers occupied during mate calling are also analyzed, and emissions from males and females result in the identification of novel putative pheromones for Z. filipendulae.  相似文献   

13.
Whereas high activities of β-glucosidase occur in homogenates of leaves of Hevea brasiliensis Muell.-Arg., this enzyme, which is capable of splitting the cyanogenic monoglucoside linamarin (linamarase), is not present in intact protoplasts prepared from the corresponding leaves. Thus, in leaves of H. brasiliensis the entire linamarase is located in the apoplasmic space. By analyzing the vacuoles obtained from leaf protoplasts isolated from mesophyll and epidermal layers of H. brasiliensis leaves, it was shown that the cyanogenic glucoside linamarin is localized exclusively in the central vacuole. Analyses of apoplasmic fluids from leaves of six other cyanogenic species showed that significant linamarase activity is present in the apoplasm of all plants tested. In contrast, no activity of any diglucosidase capable of hydrolyzing the cyanogenic diglucoside linustatin (linustatinase) could be detected in these apoplasmic fluids. As described earlier, any translocation of cyanogenic glucosides involves the interaction of monoglucosidic and diglucosidic cyanogens with the corresponding glycosidases (Selmar, 1993a, Planta 191, 191–199). Based on this, the data on the compartmentation of cyanogenic glucosides and their degrading enzymes in Hevea are discussed with respect to the complex metabolism and the transport of cyanogenic glucosides.  相似文献   

14.
Manihot esculenta (cassava) contains two cyanogenic glucosides, linamarin and lotaustralin, biosynthesized from l ‐valine and l ‐isoleucine, respectively. In this study, cDNAs encoding two uridine diphosphate glycosyltransferase (UGT) paralogs, assigned the names UGT85K4 and UGT85K5, have been isolated from cassava. The paralogs display 96% amino acid identity, and belong to a family containing cyanogenic glucoside‐specific UGTs from Sorghum bicolor and Prunus dulcis. Recombinant UGT85K4 and UGT85K5 produced in Escherichia coli were able to glucosylate acetone cyanohydrin and 2‐hydroxy‐2‐methylbutyronitrile, forming linamarin and lotaustralin. UGT85K4 and UGT85K5 show broad in vitro substrate specificity, as documented by their ability to glucosylate other hydroxynitriles, some flavonoids and simple alcohols. Immunolocalization studies indicated that UGT85K4 and UGT85K5 co‐occur with CYP79D1/D2 and CYP71E7 paralogs, which catalyze earlier steps in cyanogenic glucoside synthesis in cassava. These enzymes are all found in mesophyll and xylem parenchyma cells in the first unfolded cassava leaf. In situ PCR showed that UGT85K4 and UGT85K5 are co‐expressed with CYP79D1 and both CYP71E7 paralogs in the cortex, xylem and phloem parenchyma, and in specific cells in the endodermis of the petiole of the first unfolded leaf. Based on the data obtained, UGT85K4 and UGT85K5 are concluded to be the UGTs catalyzing in planta synthesis of cyanogenic glucosides. The localization of the biosynthetic enzymes suggests that cyanogenic glucosides may play a role in both defense reactions and in fine‐tuning nitrogen assimilation in cassava.  相似文献   

15.
Dirk Selmar 《Planta》1993,191(2):191-199
The 14C-labelled cyanogenic glucosides linustatin (diglucoside of acetone cyanohydrin) and linamarin (monoglucoside of acetone cyanohydrin), prepared by feeding [14C]valine to plants of Linum usitatissimum L., were applied to cotyledons of Hevea brasiliensis Muell.-Arg. in order to study their transport. Both [14C]-linustatin and [14C]linamarin were efficiently taken up by the cotyledons. Whereas 14C was recovered completely when [14C]linustatin was applied to the seedling, only about one-half of the radioactivity fed as [14C]linamarin could be accounted for after incubation. This observation is in agreement with the finding that apoplasmic linamarase hydrolyzes linamarin but not the related diglucoside linustatin. These data prove that, in vivo, linamarin does not occur apoplasmically and that linustatin, which is exuded from the endosperm, is taken up by the cotyledons very efficiently. Thus, these findings confirm the linustatin pathway (Selmar et al. 1988, Plant Physiol. 86, 711–716), which describes mobilization and transport of the cyanogenic glucoside linamarin, initiated by the glucosylation of linamarin to yield linustatin. When linustatin is metabolized to non-cyanogenic compounds, in Hevea this cyanogenic diglucoside is hydrolyzed by a diglucosidase which splits off both glucose molecules simultaneously as one gentiobiose moiety (Selmar et al. 1988). In contrast, [14C]linustatin, which is taken up by the cotyledon, is not metabolized but is reconverted in high amounts to the monoglucosidic [14C]linamarin, which then is temporarily stored in the cotyledons. These data demonstrate that in Hevea, besides the simultaneous diglucosidase, there must be present a further diglucosidase which is able to hydrolyze cyanogenic diglucosides sequentially by splitting off only the terminal glucose moiety from linustatin to yield linamarin. From this, it is deduced that the metabolic fate of linustatin, which is transported into the source tissues, depends on the activities of the different diglucosidases. Whereas sequential cleavage — producing linamarin — is purely a part of the process of linamarin translocation (using linustatin as the transport vehicle), simultaneous cleavage, producing acetone cyanohydrin, is part of the process of linamarin metabolization in which the nitrogen from cyanogenic glucosides is used to synthesize non-cyanogenic compounds.  相似文献   

16.
The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes L-valine as well as L-isoleucine consistent with the co-occurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k(c) value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.  相似文献   

17.
A four generation backcross breeding program was undertaken. Analysis of the levels of cyanoglucoside in Acac progeny shows that the level of cyanoglucoside (linamarin and lotaustralin) is inherited and that part of the inherited variation in cyanoglucoside levels is attributable to the existence of different Ac alleles in the parent plant. In vitro microsomal cyanoglucoside biosynthetic activity was measured in a high-level and a low-level parent plant. There was no evidence for the presence of microsomes with different qualitative properties in the two plants. The Ac locus was shown to segregate independently of the S incompatibility locus.This research was supported in part by SERC Grant GRA 95550.  相似文献   

18.
The valine/isoleucine-derived cyanogenic glycosides linamarin and lotaustralin have been isolated together with the cyclopentenoid cyanogen passibiflorin from Passiflora lutea. This is only the second report of the production of cyanogenic glycosides from more than one biosynthetic pathway in individuals of a single species.  相似文献   

19.
The common grass yellow Eurema mandarina (Pieridae, Coliadinae) widely inhabits Japan, feeds on various fabaceous plants such as silktree (Albizia julibrissin) and uses d ‐pinitol, a cyclitol omnipresent in Fabaceae, as a primary oviposition stimulant. However, E. mandarina has a clear host preference within the Fabaceae; for example, white clover (Trifolium repens) is a nonhost despite containing d ‐pinitol. The present study aims to identify plant chemicals in white clover that inhibit oviposition of E. mandarina. Females lay very few eggs on T. repens foliage and plastic plant models treated with a methanolic extract of the foliage. The foliage extract is fractionated by successive extraction with chloroform, isobutanol and water. None of these fractions induce egg‐laying responses. The aqueous fraction is further separated into four subfractions (Tr‐3‐1 to Tr‐3‐4) by column chromatography. Among these subfractions, females show high egg‐laying responses to Tr‐3‐1, which is known to contain d ‐pinitol. Interestingly, Tr‐3‐2, when mixed with Tr‐3‐1, significantly decreases egg‐laying responses, indicating that it contains oviposition deterrents. Chemical analyses reveal that two cyanogenic glucosides, linamarin and lotaustralin, are the major constituents of Tr‐3‐2. Authentic linamarin does not elicit egg‐laying responses and significantly inhibits female oviposition when mixed with Tr‐3‐1 at the natural concentration. Although these cyanogenic glucosides are reported to synergistically induce oviposition of a coliadine species Colias erate on white clover, we conclude that linamarin acts as an oviposition deterrent for E. mandarina, restricts its host range and regulates their differential host acceptance.  相似文献   

20.
The cyanogenic glucosides of four Latin American species of Acacia (Fabceae: Mimosoideae) have been isolated and characterized. Acacia atramentaria (Argentina) contains proacacipetalin, A.aroma (Argentina) linamarin and lotaustralin, A. tortuosa (Venezuela) proacacipetalin and a second presently uncharacterized glycoside, and A. globulifera (Guatemala) epiproacacipetalin which has not previously been reported as naturally occurring.  相似文献   

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